1. Field of the Invention
This invention is related in general to apparatus for aligning the microelectrodes of a voltage-clamp perfusion chamber with a target oocyte and, in particular, to an alignment mechanism suitable for automated, repeatable implementation while measuring in parallel electrophysiological responses from multiple frog oocytes in sequential experiments.
2. Description of the Related Art
As detailed in commonly-owned copending application Ser. No. 09/586,633, herein incorporated by reference in its entirety, the normal process of drug discovery involves a number of distinct stages from the initial identification of a potentially useful substance to the final step of clinical testing. Multiple screening steps are necessary to isolate substances of interest from libraries of potentially useful compounds.
The explosion of data made available from genetic research coupled with advances in chemical synthesis has produced a great demand for ever-higher screening rates to test potentially therapeutic compounds. Thus, the ability to screen compound libraries at higher and higher throughput is becoming increasingly critical in the search for new drugs, which is now a large-scale industrial activity. Accordingly, there is a growing need for integrated laboratory systems that assess large numbers of compounds quickly.
Of particular relevance to the present invention are assays conducted on Xenopus frog oocytes, which are uniquely suitable for screening of ion channels linked to a variety of diseases. Using conventional voltage clamping across the membrane of the oocyte, the voltage dependence of ion channel activity in the oocyte cell is assessed by measuring current changes produced in response to exposure to multiple test solutions. Testing of an oocyte cell under voltage-clamped conditions, a technique that is well known in the art, is carried out in batch operations in a chamber designed to support an individual oocyte being perfused with a test solution. The cell membrane is pierced with two microelectrodes of a voltage-clamp amplifier capable of recording current variations in response to voltage step changes or to the application of compounds under constant-voltage conditions.
A conventional two-electrode voltage-clamp system 10 is illustrated schematically in FIG. 1, where numerals 12 and 14 refer to a voltage-recording microelectrode and a current-passing microelectrode, respectively, inserted through the membrane 16 of an oocyte cell C. The membrane potential Vm is recorded by a unit-gain buffer amplifier 18 connected to the microelectrode 12. The membrane potential Vm is compared to a control potential Vc in a high-gain differential amplifier 20 (with gain xcexc) producing a voltage output Vxcex5 proportional to the difference xcex5 between Vm and Vc. The voltage Vxcex5 at the output of the differential amplifier 20 forces current to flow through the current-passing microelectrode 14 into the oocyte cell C, such as to drive the error xcex5 to zero and maintain the membrane voltage clamped at Vc. The circuit is completed through a ground 22 across the cell membrane, which in the schematic drawing is modeled by impedance and capacitance values Rm and Cm, respectively.
One of the main concerns in designing perfusion chambers for oocytes is the ability to isolate the oocyte cell in a stationary condition, so that it can be contacted by the voltage-clamp microelectrodes and exposed to the test solution of interest. U.S. Ser. No. 09/586,633 describes a perfusion chamber design characterized by a porous oocyte support structure with a sloped top surface that produces the automatic entrapment of the underside of the oocyte, thereby localizing the cell in a predetermined fixed position within the reach of dedicated voltage-clamp microelectrodes. The test solution is delivered continuously at the top of the chamber, above the oocyte, and withdrawn from the bottom of the chamber, below the oocyte. The porosity of the support material enables the continuous perfusion of test solution around the membrane of the oocyte, including its bottom portion that is firmly in place within the holding well. The geometry of the holding well is judiciously selected, as a function of the specific oocyte or other cell being tested, to ensure the automatic and precise placement of the cell by gravity and to optimize the pressure distribution over its membrane, thereby minimizing the probability of rupture or other damage to the cell. Once so restrained, the test cell is connected to the voltage-clamp microelectrodes and perfused with test solution in a batch operation.
The ooycyte cells C under investigation and the microelectrodes 12,14 of voltage-clamp apparatus are extremely small (typically about 1.0 mm to 1.5 mm in diameter, with the electrodes coming to a point about 0.12 mm wide). Therefore, the process of alignment of the oocyte positioned in the well of the perfusion chamber with the microelectrodes of the voltage-clamp apparatus involves a precise operation and is typically carried out by an operator with the aid of a microscope and a micromanipulator in individual workstations, performing one experiment at a time. Accordingly, it is not suitable for automated, higher-throughput, parallel-testing applications. Moreover, the configuration of many prior-art chambers often impedes direct access of the microelectrodes to the oocyte, thereby further complicating automatic insertion of the electrodes. Japanese Patent Bulletin No. 11-299496 (Fukusono et al.) describes a family of oocyte perfusion devices developed to solve this alignment problem in single-electrode voltage-clamp environments. The oocyte is placed in a conical chamber at the bottom of a cylindrical passage adapted for aligning the microelectrode with the oocyte. Because of the perfect alignment between the oocyte fixed to the chamber and the microelectrode, the tip of the electrode can be easily inserted into the oocyte without the aid of a microscope or micromanipulator. The insertion of the microelectrode can be effected manually or through a variety of mechanisms producing the linear motion of the electrode. Thus, the oocyte chamber serves the dual purpose of housing the oocyte and guiding a single microelectrode toward the oocyte.
While useful for facilitating the alignment of the microelectrode with the oocyte, the concept described by Fukusono et al. is not suitable for two-electrode voltage-clamp applications. Furthermore, it does not provide for the mechanical engagement and disengagement of the electrode from the alignment passage, which is critical for automated operation. Accordingly, the disclosed devices as well as other prior-art apparatus are not well suited for the high-throughput, electronically manipulated, automated-system needs of today""s pharmaceutical industry. This invention provides a mechanism designed to address these needs.
The primary objective of this invention is a voltage-clamp apparatus suitable for sequential testing of animal cells through successive exposures to multiple perfusion solutions in an automated, continuous system.
In particular, an objective of the invention is an automated alignment system for the correct placement of the voltage-clamp electrodes within the perfusion chamber, so that no microscope or other manually operated control device is used to guide the electrodes into the test cell.
A goal is also a microelectrode alignment system that permits the rapid change of an inoperative electrode without the need for recalibration of the guiding mechanism.
Another goal of the invention is a design particularly suitable for the testing of oocytes, especially Xenopus oocytes. 
Another objective is a voltage-clamp apparatus that can be adapted for parallel testing of multiple oocytes in a higher-throughput testing system.
Another goal is a voltage-clamp design that is suitable for implementation within an overall automated voltage-clamp and solution-delivery system.
Yet another object is a system that can be implemented using conventional voltage-clamp hardware and software, modified only to the extent necessary to meet the design parameters of the chamber and the microelectrode alignment system of the invention.
Still another goal is a method of perfusion that enables the rapid, sequential testing of multiple oocytes with multiple test solutions on a continuous sequential basis.
A final objective is a system that can be implemented economically according to the above stated criteria.
Therefore, according to these and other objectives, the present invention consists of an automated mechanism for guiding a microelectrode toward an oocyte placed in a perfusion chamber in substantial alignment with a predetermined angle of attack deemed optimal for penetrating the oocyte. According to one aspect of the invention, the final fine alignment of the microelectrode is achieved by precisely fitting the cylindrical portion of the electrode into a conforming guide collar disposed in fixed alignment with the intended direction for voltage-clamp electrode operation. The microelectrode is loosely mounted into a guide tube that is independent of the guide collar and is adapted for advancing the electrode through the guide collar to the desired position in contact with the oocyte for testing purposes, and for retrieving the electrode out of the guide collar for replacement purposes. Because the guide tube and guide collar are independently mounted, the alignment of the tip of the microelectrode is effected by its placement within the precisely aligned guide collar, irrespective of any fine misalignments in the guide tube. Accordingly, a change of microelectrode in the guide tube does not affect its final alignment in the oocyte so long as the calibration of the guide collar is not disturbed, thereby providing a mechanism for maintaining the alignment of successive microelectrodes mounted in the system without the need for recalibration.
According to another aspect of the invention, the advancement of the tip of the microelectrode into the oocyte is controlled by tracking the impedance and/or voltage measured by the electrode as it advances through the perfusion chamber. Measurable changes occur when the tip of the electrode contacts the perfusion solution and then again when it touches and penetrates the membrane of the oocyte sample, thereby providing control signals suitable for the automated positioning of the microelectrode. According to still another aspect of the invention, a xe2x80x9cvibrationxe2x80x9d (in the form of a mechanical oscillation or an electrical buzz) may be introduced in the tip of the microelectrode as it advances toward the membrane of the oocyte in order to facilitate its penetration and avoid dimpling that may cause rupture of the cell.
Because the apparatus of the invention can operate fully on an automated basis, it is suitable for use in multi-chamber parallel-testing systems wherein each chamber is used sequentially to test series of oocytes with a variety of perfusion solutions. Multiple perfusion chambers equipped with the electrode alignment mechanism of the invention can be operated independently and in parallel with greatly reduced manual operation. In essence, operator intervention is only required for changing oocytes and for maintenance, such as replacing microelectrodes periodically or after they are damaged, and for controlling the schedule of testing. By using modular components for the perfusion chamber, the guide collar structure and the guide tube structure, perfusion chambers and/or microelectrodes can be changed between tests without the need for recalibration of the electrode alignment. Therefore, series of successive tests can be carried out rapidly and precisely.
Various other purposes and advantages of the invention will become clear from its description in the specification that follows and from the novel features particularly pointed out in the appended claims. Therefore, to the accomplishment of the objectives described above, this invention consists of the features hereinafter illustrated in the drawings, fully described in the detailed description of the preferred embodiment and particularly pointed out in the claims. However, such drawings and description disclose but one of the various ways in which the invention may be practiced.